the structure of eukaryotic translation initiation · the structure of eukaryotic translation...

The Structure of Eukaryotic Translation InitiationFactor-4E from Wheat Reveals a Novel Disulfide Bond1[OA]

Arthur F. Monzingo, Simrit Dhaliwal, Anirvan Dutt-Chaudhuri, Angeline Lyon, Jennifer H. Sadow,David W. Hoffman2, Jon D. Robertus2, and Karen S. Browning2*

Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology,University of Texas, Austin, Texas 78712

Eukaryotic translation initiation factor-4E (eIF4E) recognizes and binds the m7 guanosine nucleotide at the 5# end of eukaryoticmessenger RNAs; this protein-RNA interaction is an essential step in the initiation of protein synthesis. The structure of eIF4Efrom wheat (Triticum aestivum) was investigated using a combination of x-ray crystallography and nuclear magnetic resonance(NMR) methods. The overall fold of the crystallized protein was similar to eIF4E from other species, with eight b-strands, threea-helices, and three extended loops. Surprisingly, the wild-type protein did not crystallize with m7GTP in its binding site,despite the ligand being present in solution; conformational changes in the cap-binding loops created a large cavity at the usualcap-binding site. The eIF4E crystallized in a dimeric form with one of the cap-binding loops of one monomer inserted into thecavity of the other. The protein also contained an intramolecular disulfide bridge between two cysteines (Cys) that areconserved only in plants. A Cys-to-serine mutant of wheat eIF4E, which lacked the ability to form the disulfide, crystallizedwith m7GDP in its binding pocket, with a structure similar to that of the eIF4E-cap complex of other species. NMRspectroscopy was used to show that the Cys that form the disulfide in the crystal are reduced in solution but can be induced toform the disulfide under oxidizing conditions. The observation that the disulfide-forming Cys are conserved in plants raisesthe possibility that their oxidation state may have a role in regulating protein function. NMR provided evidence that inoxidized eIF4E, the loop that is open in the ligand-free crystal dimer is relatively flexible in solution. An NMR-based bindingassay showed that the reduced wheat eIF4E, the oxidized form with the disulfide, and the Cys-to-serine mutant protein eachbind m7GTP in a similar and labile manner, with dissociation rates in the range of 20 to 100 s21.

Protein synthesis in eukaryotic cells is a highly regu-lated process that begins with the interaction of eukar-yotic translation initiation factor-4E (eIF4E) with them7GpppN cap group at the 5# end of the mRNA (forreview, see McKendrick et al., 1999; Pestova et al., 2001;Dever, 2002; Sonenberg and Dever, 2003). The eIF4Eprotein binds with its partner eIF4G to form the eIF4Fcomplex (Keiper et al., 1999; Prevot et al., 2003), whichserves as a scaffold for the assembly of initiation factorseIF4A, eIF4B, eIF3, and poly(A)-binding protein. Thisprotein-mRNA complex recruits the 40S ribosome withits attendant initiation factors (eIF1, eIF1A, eIF2, eIF5B,and eIF5) prior to scanning for the initiator AUG codon

(Asano et al., 2000; Pestova et al., 2000; Sonenberg andDever, 2003).

The role of eIF4E in translation has been extensivelystudied, particularly in mammalian and yeast (Saccha-romyces cerevisiae) systems. In mammals, protein syn-thesis can be regulated by modulating the ability ofeIF4E to interact with eIF4G (for review, see Richter andSonenberg, 2005); this regulation is carried out by agroup of repressor proteins called 4E-binding proteins(4E-BPs; Raught and Gingras, 1999; Pyronnet et al.,2001). The 4E-BPs function by mimicking eIF4G and areable to bind and sequester eIF4E, blocking formation ofeIF4F, a complex of eIF4E and eIF4G. The 4E-BPs can bephosphorylated by the FRAP/mTOR intracellular sig-naling pathway (Raught and Gingras, 1999), whichreleases eIF4E, enabling it to form a complex witheIF4G. In mammals, eIF4E is subject to direct phosphor-ylation at Ser-209 by the kinases MNK1 and MNK2,which are activated by the mitogen-activated proteinkinase signaling pathways (Fukunaga and Hunter,1997; Waskiewicz et al., 1997; Raught and Gingras,1999; Lachance et al., 2002). The C terminus of mam-malian eIF4G has an MNK1-binding site that facilitatesaccess of MNK1 to eIF4E (Pyronnet et al., 1999;Waskiewicz et al., 1999; Bellsolell et al., 2006). The effectof phosphorylation on mammalian eIF4E is still asubject of debate, with observations linking eIF4Ephosphorylation to both increased and decreasedprotein synthesis (Pyronnet et al., 1999; Naegele andMorley, 2004; Orton et al., 2004; Worch et al., 2004;

1 This work was supported by the National Institutes of Health(grant no. GM 63593 to J.D.R.), by the National Science Foundation(grant no. MCB–0214996 to K.S.B.), by the Welch Foundation (grantnos. F–1225, F–1353, and F–1333 to J.D.R., D.W.H., and K.S.B.,respectively), and by the Center for Structural Biology from theCollege of Natural Sciences.

2 These authors contributed equally to the paper.* Corresponding author; e-mail [email protected]; fax

512–471–8696.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Karen S. Browning ([email protected]).

[OA] Open Access articles can be viewed online without a sub-scription.

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Arquier et al., 2005; Reiling et al., 2005). Phosphorylationof eIF4E does not appear to be necessary for proteinsynthesis in vitro or in vivo (McKendrick et al., 2001),and the mutation of the functional equivalent of Ser-209in Drosophila melanogaster to Ala-209 is viable but doesnot have normal growth and development (Lachanceet al., 2002). It has been reported that phosphorylationalters the ability of eIF4E to bind capped mRNAs in vitro(Scheper et al., 2002); however, phosphorylation hasrecently been reported to have no effect on ligand dis-sociation rate (koff) for cap analogs in vitro (Slepenkovet al., 2006). 5# caps on longer (12 nucleotide) RNAs haveincreased affinity for both phosphorylated and unphos-phorylated eIF4E, suggesting that there may be addi-tional stabilizing interactions with eIF4E and the mRNA(Slepenkov et al., 2006).

Higher plants possess an equivalent form of eIF4Eand in addition have an isozyme form of the protein,eIF(iso)4E (Allen et al., 1992; Browning et al., 1992;Browning, 1996, 2004). Plant eIF(iso)4E is approxi-mately 50% similar in primary sequence to plant eIF4E(Allen et al., 1992; Metz et al., 1992). Furthermore,eIF(iso)4E is found in complex with an isozyme formof eIF4G, eIF(iso)4G (Lax et al., 1985; Browning et al.,1987, 1992). eIF4G and eIF(iso)4G share some similar-ity in their domains for the binding of additionalfactors eIF4A, eIF3, and poly(A)-binding protein butdiffer significantly in molecular mass, 180,000 versus82,000 kD, respectively (Browning, 1996; Metz andBrowning, 1996). Both the eIF4F (complex of eIF4E/eIF4G) and eIF(iso)4F [complex of eIF(iso)4E/eIF(iso)4G]have similar activities in supporting the initiation oftranslation in vitro (Lax et al., 1985; Browning et al.,1992). The role of the isozyme form of the eIF4E cap-binding protein in plants is not yet clear. Fluorescencequenching experiments have indicated that wheat(Triticum aestivum) eIF(iso)4F prefers hypermethylatedcap structures and mRNAs with less secondary struc-ture compared to wheat eIF4F (Balasta et al., 1993;Gallie and Browning, 2001). Plant eIF(iso)4F has alsobeen shown to localize to maize (Zea mays) mi-crotubules both in vitro and in vivo, suggesting thatlocalization of eIF(iso)4F may have a role in the trans-lation of specialized proteins within the cell (Bokroset al., 1995). Arabidopsis (Arabidopsis thaliana) eIF4Eand eIF(iso)4E mRNAs are differentially expressed(Rodriguez et al., 1998), with the eIF4E mRNA beingexpressed in all tissue types except certain root cells,while eIF(iso)4E mRNA is particularly abundantin floral organ cells and young, developing tissues(Rodriguez et al., 1998). It was also demonstrated thatArabidopsis eIF4E could functionally complementa yeast eIF4E knockout, whereas eIF(iso)4E couldnot (Rodriguez et al., 1998). Resistance to some plantviruses has been shown to reside in the genes foreIF4E or eIF(iso)4E (for review, see Kang et al., 2005b;Robaglia and Caranta, 2006). These results suggest thatalthough the biochemical properties of eIF4E andeIF(iso)4E are similar, there are differences in theirfunction in plant cells.

The structures of eIF4E in complex with mRNA capanalog from several different organisms have previouslybeen determined. The structure of yeast eIF4E wassolved by NMR methods (Matsuo et al., 1997), and themurine eIF4E (Marcotrigiano et al., 1997) and humaneIF4E (Tomoo et al., 2002, 2003) structures were solvedby x-ray crystallography. These molecules were found tohave identical topologies, with each protein containing amixed eight-stranded b-sheet and three a-helices. Ineach case, the purine ring of the cap analog was bound tothe protein using a p-p stacking interaction involvinginvariant Trp residues. A solution structure of apo-eIF4Efrom yeast was recently reported, where the protein wasfound to undergo changes in structure and internalmotions in response to ligand binding, and a structuralbasis for a coupling between eIF4G and mRNA capbinding was described (Volpon et al., 2006).

In this work, we describe the first structure of aneIF4E protein from a plant, determined using a com-bination of x-ray crystallography, NMR, and muta-tional methods. These results are part of an effort tofurther understand the functional aspects of eIF4E thatare unique to plants and provide some insight into themolecular basis of the functional differences betweeneIF4E and eIF(iso)4E.

RESULTS

Crystal Structure of the Wild-Type Wheat DN-eIF4E

The initial crystals of recombinant wheat eIF4E tookover 1 year to grow. Data were collected in house and amolecular replacement solution was obtained usingmurine eIF4E as a test model (Marcotrigiano et al.,1997). Inspection of the electron density map andseveral rounds of refinement revealed that 38 aminoacids at the N terminus of the protein were not visible.N-terminal sequence analysis of protein from dissolvedcrystals confirmed that these 38 residues had beenremoved during the prolonged crystallization period.These N-terminal amino acids are not well conservedbetween species. A similar number of amino acids werenot visible at the N terminus of the murine and hu-man forms of eIF4E (Marcotrigiano et al., 1997; Tomooet al., 2002), and NMR results have shown that theN-terminal 35 amino acids of the yeast eIF4E are flexiblein solution (Matsuo et al., 1997). To expedite additionalcrystallography work, the DNA encoding wheat eIF4Ewas truncated to produce a modified protein with38 N-terminal residues removed; this protein is calledDN-eIF4E. DN-eIF4E crystallized in about 2 weeksunder the same conditions as the proteolyzed proteinand had identical unit cell parameters.

The crystals of DN-eIF4E belong to hexagonal spacegroup P61 with the following cell constants: a 5 b 566.3 A, c 5 180.9 A. The crystallographic asymmetricunit of wheat DN-eIF4E is a dimer, exhibiting non-crystallographic symmetry along a pseudo 2-fold axis(Fig. 1). The Vm of the DN-eIF4E crystals is 2.8 A3/D.

Eukaryotic Translation Initiation Factor-4E from Wheat

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Three-Angstrom electron density maps, made withdiffraction data collected in house, showed a dramaticconformational change in the area of the expected cap-binding site at the dimer interface. However, due to thelow resolution of the map, the peptide chain could notbe unambiguously traced through this area. A crystalfreezing protocol was developed that allowed 1.85 Adata to be collected at the Brookhaven synchrotron,facilitating high resolution structure determination.Crystallographic data for DN-eIF4E are summarizedin Table I. A Ramachandran plot shows 89.6% ofresidues to be in the most favorable region, 9.1% inadditional allowed space, and 1.3% in generouslyallowed space. The refined structure includes 322 sol-vent molecules.

The overall fold and the central b-sheet regions of thedimeric protein are similar to that seen in monomericeIF4E from mammals and yeast (Marcotrigiano et al.,1997; Matsuo et al., 1997; Tomoo et al., 2002). However,

the mammalian and yeast proteins differ from thewheat DN-eIF4E in that they contain an mRNA capanalog sandwiched between two invariant Trp rings inan aromatic stack; the wild-type wheat protein crystal-lized without bound cap, despite its being present insolution. The most significant differences between theprotein structures occur in the region of the two loops,residues 53 to 65 (loop 1) and 103 to 116 (loop 2), whichhold the cap-binding Trp; these correspond to Trp-62and Trp-108 in wheat eIF4E. These loops were leftout of early models of the wheat protein and werecarefully rebuilt from a series of omit maps. In themammalian and yeast proteins, these loops form ran-dom coils that come together with the bound capsandwiched between the two Trp. Loop 2 is in closeproximity to a short helix (residues 199–203 in thewheat protein) between b-strands 7 and 8. In the wheateIF4E structure, these loops are extended away fromthe surface, opening a wide cavity between the loops

Figure 1. The backbone of the dimeric structure ofwheat eIF4E. Molecule A of the dimer is shown inred; molecule B is shown in blue. Tryptophan sidechains analogous to those previously implicated incap binding (Trp-62 and Trp-108) are shown in black,as well as Cys-113 and -151, which form a disulfidebond, are shown in yellow. Loop 1 (residues 53–65)of each monomer extends into the neighboring mol-ecule in the dimer, with Trp-62 occupying the centerof the expected cap-binding pocket.

Table I. Summary of crystallographic and structure refinement statistics for the wheat eIF4E proteinlacking residues 1 to 38 at its N terminus (DN-eIF4E) and mutant C113S of the truncated protein

Wheat DN-eIF4E Mutant C113S

Space group P61 P21

Cell constants a 5 b 5 66.34,c 5 180.85 A

a 5 36.65, b 5 58.23,c 5 39.06 A, b 5 115.8�

Resolution (A) (last shell) 50.0–1.85 (1.92–1.85) 50.0–2.25 (2.32–2.25)Rmerge (%) (last shell) 7.5 (47.7) 6.3 (14.7),I/sI. (last shell) 8.7 (3.9) 16.5 (12.4)Completeness (%) (last shell) 100.0 (100.0) 96.9 (88.9)Completeness (I/sI . 3) (%) (last shell) 81.3 (46.2) 88.4 (72.7)Unique reflections 38,354 6,939Redundancy 8.2 5.5Wavelength (A) 1.1 1.5418No. of residues 354 177No. of atoms 3,130 1,553Rworking 0.211 0.200Rfree 0.234 0.274rms deviation from ideality

Bonds 0.006 0.007Angles 1.180 1.102

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and the short helix. In the cap-bound eIF4E structures,this space is occupied by the two loops and the boundcap. In the wheat protein, loop 1 is folded as a b-looprather than random coil, and in the dimer, the loop 1 ofthe neighboring monomer is inserted into the cavity,forming a four-strand antiparallel b-sheet with theloop 1 of the first monomer. Trp-108 also interacts withresidues of the empty cap-binding pocket of the neigh-boring protein in the dimer (Fig. 1). The recently re-ported NMR structure for the yeast apo-eIF4E indicatessimilar aberrant movement of the two loops that aresupposed to bind the cap analog (Volpon et al., 2006).

A Novel Disulfide Bond

Wheat eIF4E contains four Cys residues. The wheatDN-eIF4E crystal structure showed that two of theseCys, Cys-113 and Cys-151, form a disulfide bond (Fig.2). Interestingly, this pair of Cys is conserved in allknown eIF4E proteins from higher plants, but not inanimals or yeast. For example, in mouse, the homologof Cys-151 is a Cys, but the homolog of wheat Cys-113 isAsn-107 in mouse (Fig. 3). Finding a disulfide bond in acytoplasmic protein was somewhat surprising, becausethe cytoplasm of most cells is reducing. The geometryassociated with the disulfide is nearly ideal. The f andc angles for residues 113 and 151 are both in the allowedregions of a Ramachandran plot, and the C-S-S bondangles are 105.4� and 105.3� for residues 113 and 151,respectively. The C-S-S-C dihedral angle is 294.4�.These data suggest that the bond is not strained. Fromthe crystallographic data alone, it was not clear whetherthe disulfide bond is an artifact of an aerobic purifica-tion procedure and crystallization, or if it exists undersome circumstances in the cytoplasmic protein underphysiological conditions.

Crystal Structure of the C113S Mutant of Wheat DN-eIF4E

The Cys-to-Ser mutant C113S of wheat DN-eIF4E wasprepared for the purpose of further investigating the role

of the disulfide bond in the protein structure. We wereunable to crystallize the mutant protein in the ammo-nium sulfate conditions that yielded wheat DN-eIF4Ecrystals. However, the mutant protein did crystallizeunder a different, low-salt condition. These crystals wereof space group P21 with cell constants a 5 36.7, b 5 58.2,c 5 39.1 A, and b 5 115.8�. The asymmetric unitcontained a monomer, giving a Vm of 1.9 A3/D. The2.3-A structure was solved by the molecular replace-ment method. Crystallographic data for the C113S mu-tant are summarized in Table I. A Ramachandran plotshows 89.0% of residues to be in the most favorableregion, 10.4% in additional allowed space, and 0.6% ingenerously allowed space. The refined structure in-cludes 101 solvent molecules. A ribbon drawing com-paring the wild type and C113S mutant protein is shownin Figure 4.

Interestingly, it was found that unlike the wild-typewheat eIF4E, which crystallized without the boundcap, the C113S mutant contained m7GDP in a bind-ing site that is strikingly similar to that observed incrystal structures of the murine and human eIF4E(Marcotrigiano et al., 1997; Tomoo et al., 2002). Asshown in Figure 5, the purine ring of the cap issandwiched between the rings of Trp-62 and Trp-108.Conserved Glu-109 is hydrogen bonded to the meth-ylated guanine ring. Arg-158 and Arg-163 interactwith the phosphate groups of the cap; these positivelycharged amino acids are well conserved (as either Argor Lys) in mammals, yeast, and plants (Fig. 3). Anotherremarkably conserved residue, Asp-96, forms a saltbridge with Arg-158 and is therefore likely to also beimportant for cap binding. The ring of conserved Trp-167 makes a van der Waals contact with the methylgroup of the m7GDP ligand, providing a structuralbasis for the ability of the protein to discriminatebetween GDP and m7GDP.

Interestingly, the distance between Og of Ser-113 andSg of Cys-151 in the C113S mutant protein is only 3.7 A.In the wild-type protein, where both of these residuesare Cys, the sulfur atoms would be well placed to form a

Figure 2. A section of the 2Fo-Fc electron density mapof wheat eIF4E showing the disulfide bridge betweenCys-113 and Cys-151. The map is contoured at 1.0 s.





Figure 3. Amino acid sequences of eIF4E and its isoform from higher plants aligned with human and yeast eIF4E. Sequences areshown for eIF4E and eIF(iso)4E from wheat, Arabidopsis (arabi), and Nicotiana tabacum (nicot), aligned with eIF4E from Homosapiens (human) and yeast. Amino acids that are most directly involved in binding m7GTPare boxed in red. Amino acids that areimportant for stabilizing the structure of the protein are boxed in black; these are either located in the hydrophobic core orinvolved in other tertiary interactions and are well conserved across all eukarya. Plant-specific amino acids are boxed in green;these are conserved (.95%) in plants but differ from those in other eukarya. The plant-specific amino acids include Cys-113 andCys-151, which form the disulfide linkage in the wheat eIF4E crystal structure, as well as several other surface residues. Blueboxes indicate amino acids that are .90% conserved in one isoform of plant eIF4E while being variable or conserved as anotherresidue type in eIF(iso)4E and, therefore, indicative of whether the protein belongs to the eIF4E or eIF(iso)4E family. Interestingly,most of these isoform-specific residues are relatively accessible on the protein surface; hence, there is little overlap between theset of architecturally important residues (black boxes) and the isoform-specific residues (blue boxes). Although only eightsequences are shown in the figure, 55 sequences were examined in determining which residues are conserved (Joshi et al.,2005).

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disulfide linkage with a relatively minor, but perhapsfunctionally significant, perturbation of the proteinstructure. The side chain of Ser-113 (Cys-113 in thenative protein) is located relatively close to the cap-binding site; the distance between the Cb of Ser-151 andthe ring of Trp-108 is approximately 6.1 A.

Wheat eIF4E in Solution

NMR spectroscopy is well known as a tool for pro-viding detailed information about the structure, dy-namics, and stability of proteins in solution; however, itis also an effective tool for detecting Cys that formdisulfide bonds (Sharma and Rajarathnam, 2000). Spe-cifically, the 13C NMR chemical shift of the Cb in areduced Cys is typically 28 to 32 ppm, while Cys thatare involved in disulfides have Cb chemical shifts near40 ppm. An NMR method has a significant advantageover more traditional biochemical methods for disul-fide detection, such as Ellman’s assay (Ellman, 1959),because NMR can specifically indicate which Cys in theprotein sequence are involved in a disulfide whilesimultaneously monitoring whether the protein hasremained in a folded conformation during the oxida-tion attempt. In the case of wheat eIF4E, which containsfour Cys residues, we found that Ellman’s assay couldbe used to show a decrease in the number of reducedCys under oxidizing solution conditions but did notgive an unambiguous indication regarding the pres-ence or absence of the Cys-113-Cys-151 disulfide bond.

As a first step in using NMR for disulfide bonddetection, triple-resonance methods were used to as-sign the spectrum of 13C- and 15N-enriched wheatDN-eIF4E under conditions that were not intentionallyoxidizing, in a solution of 20 mM potassium phosphateat pH 7, 100 mM KCl, and m7GTP that slightlyexceeded the 1-mM protein concentration. The pres-ence of m7GTP was found to significantly increase thestability of the protein in solution. The Cb chemicalshifts of all four Cys within eIF4E were identified in athree-dimensional HN(CO) CACB spectrum (Fig. 6).Cys-113 and Cys-151 were found to have Cb chemical

shifts of 29.2 and 29.1 ppm, respectively, indicatingthat they are not involved in a disulfide bond underthese solution conditions. The other two Cys in theprotein, Cys-99 and Cys-123, have Cb chemical shiftsof 29.4 and 32.1, indicating that they are also reduced.The Cys Cb chemical shifts were unchanged in aprotein sample that had been stored for 3 months at4�C in NMR buffer without the addition of a reducingagent such as dithiothreitol (DTT), indicating that theCys are not particularly susceptible to oxidation insolution under the conditions of the NMR experi-ments. The one-dimensional NMR spectrum of theprotein contains resonance line widths that are typicalof a 20-kD molecule, consistent with wheat DN-eIF4Ebeing a monomer in solution.

In an effort to determine whether the Cys-113-Cys-151 disulfide bond that was observed in the crystals ofwheat DN-eIF4E can be formed in solution, NMR wasused to study the protein under more oxidizing condi-tions, where 10 mM hydrogen peroxide and 0.3 M

Figure 4. Ribbon diagram comparing the structure ofthe wild type (right) and C113S mutant (left) of wheateIF4E. The Trp of the cap-binding pocket are shownin green, the m7GDP is shown in magenta and thedisulfide bond (right) and positions of the reducedCys (left) are shown in yellow.

Figure 5. A section of the 2Fo-Fc electron density map of the C113Smutant of wheat eIF4E showing the bound m7GDP. The map iscontoured at 0.8 s.





ammonium sulfate were added to the sample (thecrystals were grown in ammonium sulfate). A two-dimensional 15N-1H correlated spectrum showed littleor no chemical shift changes for the large majority ofbackbone 15N and 1H nuclei, indicating that these moreoxidizing conditions did not cause the protein to de-nature or undergo a drastic conformational change.Resonance line widths were also not significantlychanged, indicating that the protein did not dimerizein solution, as it had in the crystal. However, there was asignificant decrease in intensity (or disappearance) ofthe NMR signals of approximately 20 backbone amideprotons, mostly located in loops 1 and 2, near the cap-binding site. This decrease in NMR intensity can beattributed to an increased rate of exchange of the amideprotons with the solvent and/or line resonance linebroadening due to chemical exchange, either of whichwould be consistent with these regions of the pro-tein becoming more flexible under the more oxidizingconditions. Most significantly, a three-dimensionalHN(CO) CACB spectrum obtained under the oxidizingconditions (Fig. 6) showed a chemical shift of 41.4 ppmfor Cb of Cys-113, which is typical of a Cys involved in adisulfide bond (Sharma and Rajarathnam, 2000), pre-sumably with Cys-151. There was also a small change of0.2 ppm in the Cys-113 Cb chemical shift and slightchanges in the amide 15N and 1H chemical shifts of Cys-

113 and adjacent amino acids. The Cb resonance of Cys-151 was not detected in the HN(CO) CACB spectrum,due to the amide proton of Gly-152 being unobserved,likely due to its rapid exchange with protons of thesolvent. The chemical shift of Cb of Cys-123 wasunchanged at 32.1 ppm under the oxidizing conditions,and the Cb chemical shift of Cys-99 was not detected inthe HN(CO) CACB spectrum due to the amide protonof residue 100 being unobserved.

In summary, the NMR data show that wheatDN-eIF4E does not contain a disulfide between Cys-113 and Cys-151 under solution conditions that are notintentionally oxidizing. The NMR data also provideevidence that the Cys-113-Cys-151 disulfide can beinduced to form in solution under oxidizing condi-tions; these oxidizing conditions do not denature orinduce substantial structural changes in the protein;however, there is evidence of increased flexibility inloops of the protein near the cap-binding site.

Binding of m7GTP by Wheat eIF4E in Solution

The observation that Cys-113 and Cys-151 can form adisulfide linkage without unfolding the eIF4E protein(supported by our crystallographic as well as NMRresults), combined with the observation that these Cysare well conserved in plants, suggests the possibility

Figure 6. Sections of three-dimensionalHN(CO) CACB spectra of wheat DN-eIF4Eshowing the change in chemical shift ofthe Cb of Cys-113 upon disulfide forma-tion. Peaks indicating the 13C chemicalshifts of Ca and Cb of Cys-123 and Cys-113 are labeled. Sections A and C are froma spectrum of DN-eIF4E protein in 100 mM

KCl, 10 mM potassium phosphate at pH 7,with m7GTP in slight excess over the pro-tein concentration; sections B and D showthe spectrum when 0.3 M ammonium sul-fate and 10 mM hydrogen peroxide wereadded to the NMR sample to more closelymimic the conditions under which thecrystals were obtained. Under these latterconditions, the 13C chemical shift of Cb ofCys-113 changes from 29.2 to 41.4 ppm,indicative of its participation a disulfidebond, while the chemical shifts of Cb ofCys-123 and other 1H, 13C, and 15N nucleiundergo relatively modest changes.

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that their oxidation state may have a role in proteinfunction. As part of a test of this hypothesis, an inves-tigation of the influence of disulfide formation upon theability of the protein to bind the cap analog m7GTP wasundertaken.

Biochemical methods have previously been used tostudy the binding of capped mRNAs and cap analogsby eIF4E from mammals and yeast in numerous studies(Carberry et al., 1990, 1991, 1992; Ueda et al., 1991;Minich et al., 1994; Niedzwiecka et al., 2002; Scheperet al., 2002), while cap binding by eIF4E from plants hasbeen investigated to a lesser extent (Sha et al., 1995; Renand Goss, 1996; Ruud et al., 1998; Khan and Goss, 2004;Michon et al., 2006). A fluorescence assay has been usedto quantitatively assess the binding of m7GTP by theeIF4E protein, where quenching of Trp fluorescenceupon stacking with the purine ring of the cap is mea-sured as a function of protein and cap concentration(Carberry et al., 1989; Niedzwiecka et al., 2002). In thecase of the wheat eIF4E and DN-eIF4E, the proteinswere found to be unstable in the absence of ligand (asdetected by NMR), thus preventing the preparation ofthe cap-free protein required for the fluorescence-quenching method. At low m7GTP concentrations, theprotein concentration appeared to continuously de-crease with time as measured by fluorescence emissionor UV absorbance, perhaps due to its precipitation.Instability has also been reported for the cap-freemammalian eIF4E (Niedzwiecka et al., 2002), as wellas the tendency for the protein to aggregate in theabsence of cap (Niedzwiecka et al., 2005).

NMR spectroscopy was used as an alternative tofluorescence methods for the investigation of the equi-librium and dynamics of the eIF4E-m7GTP interaction.NMR has the advantage that it can be used withoutpreparing ligand-free samples of the protein. In addi-tion, the characteristic 1H NMR spectrum of the proteincould be monitored to maintain confidence that allof the protein was folded when ligand binding wasmeasured. Upon introducing an excess of ordinary(12C rich) m7GTP into a sample of the protein that hadbeen purified with 13C-labeled ligand, it was observedthat the pool of free ligand reached an equilibriummixture of 12C and 13C in less than 90 s, which is howlong it took to acquire a 13C NMR spectrum aftermixing. This observation immediately suggested weakligand binding and put a lower limit on koff for theprotein-ligand interaction. A two-dimensional 1H-1HNOE spectrum of a mixture of wheat DN-eIF4E andexcess m7GTP shows that separate (and broadened)resonances are observed for the free and bound ligand(Fig. 7). Cross peaks between resonances of the free andbound m7GTP indicate that a significant fraction of thefree and bound pools exchange during the 200-ms NOEmixing time (Fig. 7); this NOE spectrum is remarkablysimilar to that of the Trp repressor protein obtained witha similar concentration of excess Trp (Schmitt et al.,1995). The observation that the resonances of the proteinand free ligand have the same sign in a two-dimensionalNOE spectrum is also consistent with weak ligand

binding (Post, 2003). The 1H resonances of excess freem7GTP are broadened upon the addition of a relativelysmall (less than stoichiometric) amount of wheatDN-eIF4E protein (Fig. 8). These observations indicatethat the bound and free m7GTP are in slow exchangeon the NMR time scale. An analysis of the NMR linewidths (Lian and Roberts, 1993) was used to derive theligand dissociation rate (koff) for samples of wild-type,mutant, oxidized, and reduced eIF4E protein.

Results of the NMR-based binding assay showedthat the reduced wheat DN-eIF4E, the oxidized form ofthe protein with the Cys-113-Cys-151 disulfide, and theC113S mutant protein each weakly (and similarly) bindm7GTP. For the reduced protein, koff was found to be inthe range of 48 6 15 s21; oxidized protein with thedisulfide was found to have koff of 73 6 26 s21. TheC113S mutant that cannot form the disulfide was foundto have koff in the range of 38 6 15 s21. The stateduncertainty in each value is indicative of the estimateduncertainties in the NMR line width measurements,protein and ligand concentrations, and the variabilityin data obtained using different protein preparations. Ittherefore appears that there is only a small (1.53)difference in the dissociation rate for the cap analogm7GTP for the three forms of the wheat DN-eIF4E

Figure 7. A section of a two-dimensional 1H-1H NOE spectrum of1 mM wheat DN-eIF4E in the presence of 5 mM m7GTP with thechemical shifts of free and bound m7GTP indicated by arrows. The spec-trum was obtained at 25�C with a 200-ms NOE mixing time. Theresonances of the free m7GTP have the same sign as those ofthe protein, consistent with weak ligand binding, and the resonancesof the bound m7GTP are broadened, consistent with slow exchange onthe NMR time scale. The off-diagonal exchange peaks between the freeand bound m7GTP are striking, similar to those observed in thespectrum of the Trp repressor protein in the presence of excess Trp(Schmitt et al., 1995) in a similar example of weak ligand binding. ThisNOE spectrum was obtained using a jump-return method for suppress-ing the solvent water signal, which tends to attenuate the signals ofpeaks near 5 ppm.





protein as measured by NMR. This implies that Kd forthe binding of m7GTP by wheat DN-eIF4E is greaterthan or equal to approximately 1027

M21, which is

toward the high end of the range reported for other(nonplant) formsofeIF4E, determined using fluorescence-quenching assays (Carberry et al., 1992; Niedzwieckaet al., 2002; Khan and Goss, 2004) and stopped-flowkinetic methods (Slepenkov et al., 2006). Interestingly,our NMR-based assay indicates that koff for eIF4E fromyeast is 12 6 2 s21, which is significantly different from

the reduced, oxidized, and mutant forms of the wheatprotein and suggests yeast eIF4E binds m7GTP moretightly than the wheat eIF4E (data not shown).

A lower limit for Kd can be estimated by assuming thatkon is no more rapid than the diffusion limit (approxi-mately 108–109 s21), given that Kd is defined by koff/kon.This implies that Kd for the binding of m7GTP by wheatDN-eIF4E is approximately 1027

M21 or greater, which is

consistent with values determined for mammalianeIF4E using fluorescence-quenching assays (Carberryet al., 1989; Ren and Goss, 1996; Niedzwiecka et al., 2002)and stopped-flow kinetic methods (Slepenkov et al.,2006).

DISCUSSION

The results of this work provide the first description,to our knowledge, of a structure of a plant form of thetranslation initiation factor eIF4E. As expected, thewheat protein has much in common with the mam-malian and yeast forms of eIF4E. Approximately 55residues are conserved for clearly architectural rea-sons, forming the hydrophobic core of the protein andother essential tertiary interactions (boxed in black,Fig. 3). The residues that are most intimately involvedin binding m7GTP are also conserved across all species(boxed in red, Fig. 3). Interestingly, there are 11 resi-dues that are remarkably well conserved in plants yetdiffer in identity from the amino acids in analogousposition in the mammalian and yeast forms of eIF4E;these plant-specific residues are boxed in green inFigure 3 and include Cys-113 and Cys-151, which formthe disulfide linkage observed in the crystal structureof the wheat protein. Other plant-specific residues(Asp-51, Lys-90, Lys-107, Glu-143, Asn-173, Glu-174,and Asp-201) are located in relatively accessible posi-tions on the protein surface. This combination ofconservation plus surface accessibility suggests thatthese residues are excellent candidates for being in-volved in functional interactions involving eIF4E thatare unique to translation initiation in plants. The rolesof these plant-specific surface residues will perhaps bemade clear as additional biochemical data are accu-mulated. The presence of plant-specific eIF4E surfacefeatures should not be too surprising, because severalaspects of the initiation pathway are indeed unique toplants, including the presence of two distinct forms ofeIF4E and eIF4G (Browning, 2004).

Although the roles of the two eIF4E isoforms are notyet well understood, the presence of two significantlydivergent forms of the protein in plants strongly sug-gests some isoform-specific function. It has been re-ported that a knockout of the single eIF(iso)4E gene inArabidopsis grows normally, although the amount ofeIF4E is increased in the mutants (Duprat et al., 2002),suggesting that the two forms have overlapping func-tions. Perhaps more interestingly, mutants (both natu-rally occurring and induced) for both eIF4E andeIF(iso)4E are resistant to infection by a number of

Figure 8. A, Sections of a one-dimensional 1H NMR spectrum of amixture of 0.2 mM m7GTP and the chemical shift reference standardDSS in the absence of protein. B, Sections of the 1H NMR spectrum of asample containing 0.25 mM m7GTP and a less-than-stoichiometricamount (0.03 mM) of the wheat DN-eIF4E protein, showing that thewheat DN-eIF4E broadens the methyl peak of the free m7GTP, asexpected in the case of slow exchange on the NMR time scale. Thewidth of the DSS resonance shows relatively little change upon theaddition of the protein. C, A representative graph showing thatthe width of the free m7GTP methyl resonance varies with proteinand ligand concentrations, consistent with labile ligand binding andthe model of slow exchange on the NMR timescale. [Protein] and[Ligand] refer to the total concentrations of wheat DN-eIF4E proteinand m7GTP, respectively. Dn1/2 is defined as the width of the m7GTPmethyl resonance at half height minus the width of the DSS resonance.

Monzingo et al.




plant viruses, particularly the potyviruses, suggestingthat these plant viruses exploit the eIF4E/eIF(iso)4Emachinery to their benefit (for review, see Robaglia andCaranta, 2006). The resistance phenotype arises fromdisruption of the direct interaction between the viralprotein (VPg) linked to the 5# end of the potyviral RNAand eIF4E or eIF(iso)4E (Leonard et al., 2004; Michonet al., 2006). However, the viruses have counterednatural resistance with mutations in the VPg (Kelleret al., 1998; Redondo et al., 2001; Moury et al., 2004).Naturally occurring potyvirus resistance genes havebeen shown to reside in the genes for eIF4E or eIF(iso)4E in lettuce (Latuca sativa) mo1 (eIF4E; Nicaiseet al., 2003); pepper (Capsicum annuum) pvr1 (eIF4E) andpvr6 [eIF(iso)4E; Ruffel et al., 2002, 2006; Kang et al.,2005a]; tomato (Lycopersicon esculentum) pot-1 (eIF4E;Ruffel et al., 2005); pea (Pisum sativum) sbm1 (eIF4E; Gaoet al., 2004b) and sbm2 [eIF(iso)4E; Gao et al., 2004a];and Arabidopsis lsm1 [eIF(iso)4E; Duprat et al., 2002;Lellis et al., 2002; Sato et al., 2005] and cum1 (eIF4E;Yoshii et al., 2004; Sato et al., 2005). The barley (Hordeumvulgare) rym4/5/6 resistance genes to various strains ofthe bymoviruses, Barley yellow mosaic virus and Barleymild mosaic virus, were also found to be mutations in theeIF4E gene (Kanyuka et al., 2005; Stein et al., 2005).

The exact role of eIF4E and eIF(iso)4E in virusresistance is not clear. A number of hypotheses havebeen proposed for the eIF4E/eIF(iso)4E-mediated vi-ral resistance. These include disruption of host trans-lation initiation (Lellis et al., 2002), stabilization of theviral RNA from degradation (Lellis et al., 2002), dis-ruption of trafficking of the viral RNA (Lellis et al.,2002), interference with viral replication (Robagliaand Caranta, 2006), sequestration of initiation factors

(Kanyuka et al., 2005), and interference with cell to cellmovement (Gao et al., 2004b). In the case of resistanceof Pepper veinal mottle virus, both the pvr2 (eIF4E) andpvr6 [eIF(iso)4E] mutations are required for resistance(Ruffel et al., 2006). The role of the interaction of theVPg and eIF4E/eIF(iso)4E in the viral infection/repli-cation cycle is far from clear (Robaglia and Caranta,2006).

A number of structural models for plant eIF4E havebeen proposed based on the murine or human structure(Marcotrigiano et al., 1997; Tomoo et al., 2002) to showthe positions of these naturally occurring mutations inplant eIF4E (Gao et al., 2004b; Kang et al., 2005a;Kanyuka et al., 2005; Ruffel et al., 2005; Stein et al.,2005; Albar et al., 2006; Robaglia and Caranta, 2006).These models predicted that the mutations are largelyon the surface of eIF4E and some are near the cap-binding pocket. However, very few of the naturallyoccurring mutations have been shown to affect capbinding per se (Kang et al., 2005a). The residues knownto be involved in potyvirus or bymovirus resistance areshown at the comparable position in wheat eIF4E(see Fig. 9) and are numbered to be consistent withthe wheat eIF4E sequence in Figure 3. The naturally oc-curring resistance mutations in eIF4E for potyvirusesin tomato (red), lettuce (blue), pepper (orange), andpea (green) and the bymovirus resistance mutationsin eIF4E (yellow) are modeled in Figure 9. Most ofthe resistance genes carry more than one amino acidmutation, although the lettuce mo12 is a single muta-tion (S55P; Nicaise et al., 2003), and a single muta-tion (Q161H) generated by ethyl methanesulfonatemutagenesis confers bymovirus resistance (Kanyukaet al., 2005). These single mutations suggest that by

Figure 9. Plant virus resistance mutationsmodeled on the wheat eIF4E structure. A,The residues implicated in plant potyvirusresistance are shaded as follows on row 1,tomato (red; Ruffel et al., 2005), lettuce(light blue; Nicaise et al., 2003), pea(green; Gao et al., 2004b), and pepper(orange; Ruffel et al., 2002; Kang et al.,2005a). The bymovirus resistance residuesare in yellow on row 2 (Stein et al., 2005;Kanyuka et al., 2005). Residues implicatedin more than one species are shaded indark blue on row 3. The view on the leftshows m7GDP in the binding pocket. Theview on the right is rotated 90� to the rightabout the vertical axis. B, The mammalianeIF4G peptide (purple; Marcotrigianoet al., 1999) is modeled on the wheatstructure to show its relative position to theall potyvirus resistance mutations (shadeddark blue).





disrupting a single contact, the VPg interaction iscompromised. Generally, two regions of the eIF4E pro-tein are targeted in virus resistance, one near the cap-binding site, but not directly part of it, and anotherrotated 90� from the cap-binding pocket (see Fig. 9).These two regions on different facets of the eIF4Emolecule suggest that the VPg may have two bindingsites for optimal interaction. The single amino acidmutations at different sites of eIF4E suggest that bind-ing at both sites is necessary for viral infection. Onlyfour of the mutations would appear to have any poten-tial direct effect on cap binding. The mutation at posi-tion G94 in tomato (G94R), lettuce (G94H), and pepper(G94R) points toward the phosphate, and the morehydrophilic substitutions (Arg or His) could formhydrogen bonds with the phosphate backbone of themRNA. Pea N156K and barley Q161K also could inter-act with the mRNA phosphate backbone. Only barleyE109G, where the Glu forms a direct hydrogen bondwith the guanine ring, would have a direct effect on capbinding. The precise contact points between the VPgand the eIF4E/eIF(iso)4E appear to be optimized foreach virus/host pair by coevolution, because theyoccur at different positions (compare potyvirus andbymovirus in Fig. 9). A recent study of the VPg-eIF4Einteraction in vitro indicated that the binding ofthe potyvirus lettuce mosaic virus VPg and m7GDP toeIF4E were not mutually exclusive; that is, both couldbe bound, but binding of one ligand did reduce theaffinity for the other about 15-fold (Michon et al., 2006).In addition, VPg increased the binding of a peptidecontaining the eIF4E-binding domain of eIF4G to eIF4E.This result suggests that the VPg is able to manipulatethe eIF4E/eIF4G interaction (Michon et al., 2006). How-ever, the eIF4G-binding domain of plant eIF4E (Fig. 9C,shown in purple) is at a considerable distance from theresistance mutations, indicating that in the case of theseparticular host/virus pairs, resistance is likely not adirect result of the eIF4G interaction but more likelydue to a conformational change in eIF4E upon bindingof VPg. Mutations in eIF4G and eIF(iso)4G have alsobeen reported to confer resistance to various viruses inArabidopsis and rice (Oryza sativa), respectively (Yoshiiet al., 2004; Albar et al., 2006). Thus, it appears that avariety of methods for the virus to interact with thehost’s translational apparatus have evolved, and thehosts have responded by coevolving mutations forresistance within these genes. The structure of a planteIF4E will be very valuable in elucidating the mecha-nisms for plant virus resistance and potentially for thedesign of virus resistant plants.

The structure of wheat eIF4E described in this work,when combined with a comparative sequence analysis,provides information that can be used to gain someinsight into the features that distinguish the two isoformsthat occur in plants. Thirteen residues are .90% con-served as one residue type in one isoform of the eIF4Eprotein, while being variable or conserved as a differentresidue type in the other isoform (Table II). Interestingly,most of these isoform-specific residues are located in

relatively accessible positions on the protein surface. Fiveof these residues (53, 55, 59, 103, and 112) are located inloops relatively near the site of the bound m7GDP nu-cleotide, suggesting that plant eIF4E and eIF(iso)4E maydiffer in their interactions with the mRNA cap or a regionof the mRNA near the cap. Several other isoform-specificresidues (83, 127, 128, 134, 183, and 193) are also locatedon protein surface, though relatively far from the cap-binding site. These isoform-specific surface residues(boxed in blue in Fig. 3) are excellent candidates forbeing the key sites that are central to isoform-specificfunction. Interestingly, only two of these isoform-specificresidues correspond to the virus resistance mutations(the mo12 single mutation, A55P, and one in rym6, P53S),suggesting that virus resistance and isoform-specificfunctions are not directly linked.

The role of the disulfide bond in plant eIF4E has yetto be fully elucidated. The redox state of the plant cellcan vary in response to a number of events, includinglight or environmental stresses (heat, drought, patho-gens, etc.), and plants use redox as a mechanism forregulation of photosynthesis, seed development, andgermination (Buchanan and Balmer, 2005). The appar-ently strong conservation of Cys-113 and Cys-151 inplants, along with their close proximity in the proteinstructure and observed ability to form a disulfidelinkage, together raise the possibility that disulfideformation may have some biological function, such asa regulatory role. It is very intriguing to speculate thatredox may be a method unique to plants for regulatingthe initiation of protein synthesis.

Perhaps the simplest hypothesis would be thatdisulfide formation influences the ability of eIF4E to

Table II. Summary of the isoform-specific amino acids that areindicative of whether a plant protein belongs to the eIF4E oreIF(iso)4E family

These residues are .95% conserved as one residue type in oneisoform while being .95% conserved as a different residue type in theother isoform of eIF4E. These isoform-specific residues are excellentcandidates for being participants in intermolecular interactions thatmay functionally distinguish eIF4E from eIF(iso)4E. A set of 55 eIF4Eand eIF(iso)4E sequences from 23 diverse plant species was examined(Joshi et al., 2005). Residues are numbered to be consistent with thewheat eIF4E sequence in Figure 3.

Residue No. Identity in Plant eIF4E Identity in Plant eIF(iso)4E

53 Pro Glu55 Variable Lys59 Gln/Lys Gly83 Asn Asp/Glu94 Gly Asn/Ser99 Cys Leu

103 Lys Gly112 Ile/Val Glu127 Lys Variable128 Ser Leu/Phe134 His/Tyr Glu183 Gln Lys193 Ser/Thr Lys

Monzingo et al.




bind capped mRNA, although this is not supported bythe results of this work, where modest difference(1.53) was observed in the abilities of the reducedand oxidized wheat eIF4E to bind m7GTP. However,eIF4E in vivo is part of a larger multiprotein complex,and binding of m7GTP by eIF4E is not an ideal reporterof the ability of the eIF4E-eIF4G complex to bind acapped mRNA. As a specific example, it has beenshown that eIF4E binding of 5#mRNA cap is signifi-cantly stabilized by domains of eIF4G (Von der Haaret al., 2006). So the possibility does remain that theoxidation state of these conserved Cys may modulatesome aspect of eIF4E function in vivo by affecting itsability to bind capped mRNA, as well as form com-plexes with eIF4G, other eIF4E-binding proteins, ormembers of the translational machinery. Further workwill be necessary to establish a role, if any, of thedisulfide bond present in plant eIF4E/eIF(iso)4E in theregulation of plant protein synthesis.

MATERIALS AND METHODS

Preparation of Full-Length Wheat eIF4E

A cDNA expression clone based on Z12616 (Metz et al., 1992) for wheat

(Triticum aestivum) eIF4E was constructed by DNA amplification and inserted

into plasmid pET22b (Novagen). This clone differed from Z12616 in that the

DNA sequence in the first approximately 60 nucleotides was modified to

reduce the high GC content, but maintained the correct amino acid sequence.

The expression clone was transformed into BL21pLysS (Novagen) cells for

expression. Procedures for expression and purification of wheat eIF4E for

crystallization were similar to those described (Van Heerden and Browning,

1994; Mayberry, et al., 2007). Briefly, a 2.5-L culture was grown to an O.D. of

approximately 0.8 at 37�C. Expression of eIF4E was induced by the addition of

0.64 mM isopropylthio-b-galactoside and grown for an additional 2 h at 37�C.

The cells were harvested and resuspended in 20 mL of buffer (20 mM HEPES-

KOH, pH 7.6, 0.1 mM EDTA, 2.0 mM DTT, 10% glycerol, 50 mM KCl, 100 mM

GTP) containing two dissolved Mini-Protease Inhibitor tablets (Boerhinger-

Mannheim). The cells were disrupted by sonication and centrifuged for 1 h in

a 60Ti rotor at 47,500 rpm. The supernatant was applied to a 3-mL m7GTP-

Sepharose (Pharmacia) column equilibrated in 20 mM HEPES-KOH, pH 7.6,

0.1 mM EDTA, 2.0 mM DTT, 10% glycerol, and 50 mM KCl. The column was

washed with equilibration buffer until the O.D. dropped to baseline. The

column was then washed with 25 mL of 20 mM HEPES-KOH, pH 7.6, 2.0 mM

DTT, and 0.2 mM GTP. The GTP helps removes a protein contaminant that

copurifies with eIF4E (identified as DNA J by protein sequence analysis; data

not shown). The eIF4E protein was eluted from the affinity column with 25 mL

of 20 mM HEPES-KOH, pH 7.6, 2.0 mM DTT, and 0.2 mM m7GTP or m7GDP.

Fractions containing the highest amount of protein were pooled and concen-

trated to approximately 1 to 2 mg/mL for crystallization trials.

N-terminal protein sequence analysis of the crystallized eIF4E was done at

the Protein Microanalysis Facility at the University of Texas at Austin.

Preparation of Truncated and Mutant Wheat eIF4E

A cDNA expression clone for wheat eIF4E truncating amino acids 1 to 38

(referred to as DN-eIF4E) was prepared by DNA amplification and placed in

plasmid pET15b (Novagen); the C113S variant of DN-eIF4E was also prepared

using DNA amplification. The DN-eIF4E protein was expressed and purified

essentially the same as the full-length protein. The purification of the C113S

mutant DN-eIF4E protein used 20 mM HEPES-KOH, pH 7.6, 0.1 mM EDTA,

2.0 mM DTT, 10% glycerol, and 50 mM KCl for washing of m7GTP-Sepharose

column, and the protein was eluted with the same buffer containing 0.2 mM

m7GDP. The eluted protein was concentrated to approximately 1 to 5 mg/mL for

crystallization. Proteins used in NMR experiments were prepared by washing

the affinity column with a buffer of 10 mM phosphate, pH 7, and 100 mM KCl

prior to elution with 0.2 mM m7GTP in the same phosphate buffer. Protein

samples enriched in 15N and/or 13C were prepared as above, but with M9

minimal medium containing 0.5 g/L 15N ammonium chloride and/or 2 g/L 13C

Glc (Cambridge Isotope Laboratories) as the sources of nitrogen and/or carbon.

Crystallization and Data Collection

Wheat wild-type eIF4E, in elution buffer containing 0.2 mM m7GTP, was

crystallized by the hanging drop method at 4�C from 1.8 to 2.4 M ammonium

sulfate in 0.1 M HEPES or Tris-HCl buffer, pH 7.5 to 8.0. Prior to collecting cold

stream data, a wild-type crystal was dipped for 1 to 5 s into a solution of 50%

saturated sorbitol in artificial mother liquor (2 M ammonium sulfate, 25 mM

Tris-HCl, pH 8.0).

C113S mutant DN-eIF4E in elution buffer containing 0.2 mM m7GDP was

crystallized at 4�C using the hanging drop method from 28% (w/v) PEG4000,

0.1 M HEPES, pH 7.0, 20 mM phenol. Prior to data collection, the crystal was

treated with cryoprotectant by transferring to artificial mother liquor (35%

PEG4000, 0.1 M HEPES, pH 7.0) for 1 to 5 s. Following treatment with cryo-

protectant, a wild-type or mutant crystal mounted in a cryoloop (Hampton

Research) was then frozen by dipping into liquid nitrogen and placed in the

cold stream on the goniostat.

Data from a C113S mutant crystal and preliminary wild-type crystal were

collected in-house at 100 K on a Rigaku RAXIS IV image plate detector with a

Rigaku RU-H3R rotating copper anode generator (Rigaku/MSC) operated at

50 kV and 100 mA. Data from a DN-eIF4E crystal were collected at 100 K on an

ADSC Quantum 4 CCD detector at beamline X12-B of the National Synchro-

tron Light Source, Brookhaven National Laboratory. Raw data were reduced

using the programs DENZO and SCALEPACK from the HKL suite

(Otwinowski and Minor, 1997).

Crystal Structure Determination and Analysis

Molecular replacement with both wild-type and mutant data was carried

out using murine eIF4E (Protein Data Bank ID 1EJ1; Marcotrigiano et al., 1997)

as a model; the wheat and murine proteins have approximately 40% sequence

identity. The molecular replacement and initial refinement with approxi-

mately 3 A wild-type data collected in house were carried out using the

Evolutionary Protein Molecular Replacement program (Kissinger et al., 1999)

and X-PLOR (Brunger, 1992). Initial phasing and electron density map

improvement with high resolution DN-eIF4E data were generated from the

molecular replacement solution using the program ARP/wARP (Lamzin and

Wilson, 1993; Perrakis et al., 1997). Molecular replacement with the C113S data

was done with the program MOLREP (Vagin and Teplyakov, 1997).

Following manual adjustment using the program O (Jones et al., 1991), the

model was refined with the Crystallography and NMR system (CNS, version

1.0) suite using the slow-cooling protocol (Brunger et al., 1998). There were

several rounds of refinement followed by rebuilding of the model. To facilitate

manual rebuilding of the model, a difference map and a 2Fo-Fc map, SIGMAA

weighted to eliminate bias from the model (Read, 1986), were prepared. Five

percent of the diffraction data were set aside throughout refinement for cross

validation (Brunger, 1993). Geometry and stereochemistry of the models were

analyzed throughout building and refinement using PROCHECK (Laskowski,

1993). For the purpose of building bound waters into the model, CNS was

used to select solvent peaks. Candidates were positive peaks on a difference

map 3.5 SDs above the mean and within 3.5 A of a protein nitrogen or oxygen

atom. O was used to manually view and accept water sites. Computations

were done on a Gateway Select SB computer. Model visualization and rebuild-

ing was done on a Silicon Graphics Indy computer.

Pictures and drawings were constructed using MOLSCRIPT (Kraulis,

1991), BOBSCRIPT (Esnouf, 1999), and PyMOL (Delano Scientific). Atomic

coordinates for the dimeric wheat DN-eIF4E (2IDR) and its monomeric C113S

mutant (2IDV) have been deposited in the Protein Data Bank.

NMR Spectroscopy

NMR spectra were recorded at 25�C using a 500-MHz Varian Inova

spectrometer equipped with a triple-resonance cryogenic probe and z axis

pulsed field gradient. NMR samples typically contained 1.0 mM of DN-eIF4E

protein in 90% water/10% D2O plus 100 mM KCl, a buffer of 10 mM potassium

phosphate at pH 7, and m7GTP in slight excess over the protein concentration.

Backbone 1H, 13C, and 15N resonance assignments were obtained using three-

dimensional HNCA, HNCACB, HN(CO) CACB, HNCO, and HACACBCO





spectra (Grzesiek et al., 1992; Kay, 1993; Muhandiram and Kay, 1994). Side

chain resonance assignments were obtained using three-dimensional 15N-1H-1H

HSQC-TOCSY and 13C-1H-1H HCCH-TOCSY spectra (Kay et al., 1993). Data

were processed using NMR-Pipe (Delaglio et al., 1995) and visualized using

Sparky (Goddard and Kneller, 2006). 1H, 15N, and 13C chemical shifts were

referenced as recommended (Wishart et al., 1995), with proton chemical shifts

relative to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), and 13C and15N reference frequencies determined by multiplying the 1H reference fre-

quency by 0.251449530 and 0.101329118, respectively.

The off rate for the interaction between wheat DN-eIF4E and m7GTP was

derived from the width of the m7GTP methyl resonance measured at various

protein and ligand concentrations. Data were obtained in NMR buffer at 25�C

and fit to the equation: pDy 2 pDyo 5 koff ([P]/[L] 2 [P]), where Dy is the width

at half height of the methyl resonance of m7GTP, Dyo is the width in the

absence of protein, [P] is the total protein concentration, and [L] is the total

ligand concentration (Lian and Roberts, 1993); this analysis assumes slow

exchange on the NMR time scale and free ligand concentrations that signif-

icantly exceed Kd. In mixtures used for NMR line width measurements,

protein concentrations were typically 0.02 to 0.06 mM, as measured by

Bradford assay (Bradford, 1976), and ligand concentrations were typically in

the range of 0.2 to 1.0 mM; under these conditions, broadening of the 1H

resonances of the free m7GTP was typically in the range of 1 to 10 Hz due to

chemical exchange. The 0.0 ppm resonance of DSS was used as an internal

NMR line width standard to control for variations in shimming. NMR-based

ligand binding assays were carried out in (at least) triplicate.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number Z12616.

ACKNOWLEDGMENTS

We thank Kelley Ruud Nitka and Daniel Hirschhorn for technical assis-

tance in the initial wheat eIF4E preparation and crystallization, and Patricia

Murphy for technical assistance in the preparation of wheat DN-eIF4E. We

thank Alexander Taylor and John Hart for data collection at beamline X12-B

at the National Synchrotron Light Source, Brookhaven National Laboratory;

support for the beamline comes principally from the Offices of Biological and

Environmental Research and of Basic Energy Sciences of the U.S. Department

of Energy and from the National Center for Research Resources of the

National Institutes of Health.

Received November 15, 2006; accepted February 21, 2007; published February

23, 2007.

LITERATURE CITED

Albar L, Bangratz-Reyser M, Hebrard E, Ndjiondjop MN, Jones M,

Ghesquiere A (2006) Mutations in the eIF(iso)4G translation initiation

factor confer high resistance of rice to rice yellow mottle virus. Plant J 47:

417–426

Allen ML, Metz AM, Timmer RT, Rhoads RE, Browning KS (1992) Isolation

and sequence of the cDNAs encoding the subunits of the isozyme form of

wheat protein synthesis initiation factor 4F. J Biol Chem 267: 23232–23236

Arquier N, Bourouis M, Colombani J, Leopold P (2005) Drosophila Lk6 kinase

controls phosphorylation of eukaryotic translation initiation factor 4E and

promotes normal growth and development. Curr Biol 15: 19–23

Asano K, Clayton J, Shalev A, Hinnebusch AG (2000) A multifactor

complex of eukaryotic initiation factors, eIE1, eIF2, eIF3, eIF5, and

initiator tRNAMet is an important translation initiation intermediate in

vivo. Genes Dev 14: 2534–2546

Balasta ML, Carberry SE, Friedland DE, Perez RA, Goss DJ (1993)

Characterization of the ATP-dependent binding of wheat germ protein

synthesis initiation factors eIF-(iso)4F and eIF-4A to mRNA. J Biol Chem

268: 18599–18603

Bellsolell L, Cho-Park PF, Poulin F, Sonenberg N, Burley SK (2006) Two

structurally atypical HEAT domains in the C-terminal portion of human

eIF4G support binding to eIF4A and Mnk1. Structure 14: 913–923

Bokros CL, Hugdahl JD, Kim H-H, Hanesworth VR, Van Heerden A,

Browning KS, Morejohn LC (1995) Function of the p86 subunit of

eukaryotic initiation factor (iso)4F as a microtubule-associated protein

in plant cells. Proc Natl Acad Sci USA 92: 7120–7124

Bradford MM (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilizing the principle of protein-dye

binding. Anal Biochem 72: 248–254

Browning KS (1996) The plant translational apparatus. Plant Mol Biol 32:

107–144

Browning KS (2004) Plant translation initiation factors: it is not easy to be

green. Biochem Soc Trans 32: 589–591

Browning KS, Lax SR, Ravel JM (1987) Identification of two messenger

RNA cap binding proteins in wheat germ: evidence that the 28-kDa

subunit of eIF-4B and the 26-kDa subunit of eIF-4F are antigenically

distinct polypeptides. J Biol Chem 262: 11228–11232

Browning KS, Webster C, Roberts JKM, Ravel JM (1992) Identification of

an isozyme form of protein synthesis initiation factor 4f in plants. J Biol

Chem 267: 10096–10100

Brunger AT (1992) X-PLOR Version 3.1: A System for X-Ray Crystallography

and NMR. Yale University Press, New Haven, CT

Brunger AT (1993) Assessment of phase accuracy by cross validation: the

free R value. Methods and applications. Acta Crystallogr D Biol

Crystallogr 49: 24–36

Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-

Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, et al

(1998) Crystallography & NMR system: a new software suite for macro-

molecular structure determination. Acta Crystallogr D Biol Crystallogr

54: 905–921

Buchanan BB, Balmer Y (2005) Redox regulation: a broadening horizon.

Annu Rev Plant Biol 56: 187–220

Carberry SE, Darzynkiewicz E, Goss DJ (1991) A comparison of the

binding of methylated cap analogues to wheat germ protein synthesis

initiation factors 4F and (iso)4F. Biochemistry 30: 1624–1627

Carberry SE, Darzynkiewicz E, Stepinski J, Tahara SM, Rhoads RE, Goss

DJ (1990) A spectroscopic study of the binding of N-7-substituted cap

analogues to human protein synthesis initiation factor 4E. Biochemistry

29: 3337–3341

Carberry SE, Friedland DE, Rhoads RE, Goss DJ (1992) Binding of pro-

tein synthesis initiation factor 4E to oligoribonucleotides: effects of cap

accessibility and secondary structure. Biochemistry 31: 1427–1432

Carberry SE, Rhoads RE, Goss DJ (1989) A spectroscopic study of the

binding of m7GTP and m7GpppG to human protein synthesis initiation

factor 4E. Biochemistry 28: 8078–8083

Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995)

NMRPipe: a multidimensional spectral processing system based on

UNIX pipes. J Biomol NMR 6: 277–293

Dever TE (2002) Gene-specific regulation by general translation factors.

Cell 108: 545–556

Duprat A, Caranta C, Revers F, Menand B, Browning KS, Robaglia C

(2002) The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable

for plant growth but required for susceptibility to potyviruses. Plant J

32: 927–934

Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82: 70–77

Esnouf RM (1999) Further additions to MolScript version 1.4, including

reading and contouring of electron-density maps. Acta Crystallogr D

Biol Crystallogr 55: 938–940

Fukunaga R, Hunter T (1997) MNK1, a new MAP kinase-activated protein

kinase, isolated by a novel expression screening method for identifying

protein kinase substrates. EMBO J 16: 1921–1933

Gallie DR, Browning KS (2001) eIF4G functionally differs from eIFiso4G in

promoting internal initiation, cap-independent translation, and trans-

lation of structured mRNAs. J Biol Chem 276: 36951–36960

Gao Z, Eyers S, Thomas C, Ellis N, Maule AJ (2004a) Identification of

markers tightly linked to sbm recessive genes for resistance to Pea seed-

borne mosaic virus. Theor Appl Genet 109: 488–494

Gao Z, Johansen E, Eyers S, Thomas CL, Noel Ellis TH, Maule AJ (2004b)

The potyvirus recessive resistance gene, sbm1, identifies a novel role for

translation initiation factor eIF4E in cell-to-cell trafficking. Plant J 40:

376–385

Goddard TD, Kneller DG (2006) SPARKY 3. University of California,

San Francisco. http://www.cgl.ucsf.edu/home/sparky/

Grzesiek S, Dobeli H, Gentz R, Garotta G, Labhardt AM, Bax A (1992) 1H,13C, and 15N NMR backbone assignments and secondary structure of

human interferon-gamma. Biochemistry 31: 8180–8190

Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for

building protein models in electron density maps and the location of

errors in these models. Acta Crystallogr A 47: 110–119

Monzingo et al.




Joshi B, Lee K, Maeder DL, Jagus R (2005) Phylogenetic analysis of eIF4E-

family members 2. BMC Evol Biol 5: 48

Kang BC, Yeam I, Frantz JD, Murphy JF, Jahn MM (2005a) The pvr1 locus

in Capsicum encodes a translation initiation factor eIF4E that interacts

with tobacco etch virus VPg. Plant J 42: 392–405

Kang BC, Yeam I, Jahn MM (2005b) Genetics of plant virus resistance.

Annu Rev Phytopathol 43: 581–621

Kanyuka K, Druka A, Caldwell DG, Tymon A, McCallum N, Waugh R,

Adams MJ (2005) Evidence that the recessive bymovirus resistance

locus rym4 in barley corresponds to the eukaryotic translation initiation

factor 4E gene. Mol Plant Pathol 6: 449–458

Kay LE (1993) Pulsed-field gradient-enhanced three-dimensional NMR

experiment for correlating 13Ca./b, 13C’, and 1Ha chemical shifts in

uniformly 13C-labeled proteins dissolved in water. J Am Chem Soc 115:

2055–2057

Kay LE, Xu GY, Singer AU, Muhandiram DR, Forman-Kay JD (1993) A

gradient-enhanced HCCH-TOCSY experiment for recording side-chain1H and 13C correlations in H2O samples of proteins. J Magn Reson B 101:

333–337

Keiper BD, Gan WN, Rhoads RE (1999) Protein synthesis initiation factor

4G. Int J Biochem Cell Biol 31: 37–41

Keller KE, Johansen IE, Martin RR, Hampton RO (1998) Potyvirus

genome-linked protein (VPg) determines pea seed-borne mosaic virus

pathotype-specific virulence in Pisum sativum. Mol Plant Microbe

Interact 11: 124–130

Khan MA, Goss DJ (2004) Phosphorylation states of translational initiation

factors affect mRNA cap binding in wheat. Biochemistry 43: 9092–9097

Kissinger CR, Gehlhaar DK, Fogel DB (1999) Rapid automated molecular

replacement by evolutionary search. Acta Crystallogr D Biol Crystallogr

55: 484–491

Kraulis PJ (1991) MOLSCRIPT: a program to produce both detailed and

schematic plots of protein structures. J Appl Crystallogr 24: 946–950

Lachance PED, Miron M, Raught B, Sonenberg N, Lasko P (2002)

Phosphorylation of eukaryotic translation initiation factor 4E is critical

for growth. Mol Cell Biol 22: 1656–1663

Lamzin VS, Wilson KS (1993) Automated refinement of protein models.

Acta Crystallogr D Biol Crystallogr 49: 129–147

Laskowski RA (1993) PROCHECK: a program to check the stereochemical

quality of protein structures. J Appl Crystallogr 26: 283–291

Lax S, Fritz W, Browning K, Ravel J (1985) Isolation and characterization of

factors from wheat germ that exhibit eukaryotic initiation factor 4b

activity and overcome 7-methylguanosine 5#triphosphate inhibition of

polypeptide synthesis. Proc Natl Acad Sci USA 82: 330–333

Lellis AD, Kasschau KD, Whitham SA, Carrington JC (2002) Loss-of-

susceptibility mutants of Arabidopsis thaliana reveal an essential role for

eIF(iso)4E during potyvirus infection. Curr Biol 12: 1046–1051

Leonard S, Viel C, Beauchemin C, Daigneault N, Fortin MG, Laliberte JF

(2004) Interaction of VPg-Pro of turnip mosaic virus with the translation

initiation factor 4E and the poly(A)-binding protein in planta. J Gen

Virol 85: 1055–1063

Lian L-Y, Roberts GCK (1993) NMR of Macromolecules: A Practical

Approach. Oxford University Press, New York, pp 153–182

Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK (1997) Co-crystal

structure of the messenger RNA 5# cap-binding protein (eIF4E) bound to

7-methyl-GDP. Cell 89: 951–961

Marcotrigiano J, Gingras AC, Sonenberg N, Burley SK (1999) Cap-

dependent translation initiation in eukaryotes is regulated by a molec-

ular mimic of elF4G. Mol Cell 3: 707–716

Matsuo H, Li HJ, McGuire AM, Fletcher CM, Gingras AC, Sonenberg N,

Wagner G (1997) Structure of translation factor eIF4E bound to m7GDP

and interaction with 4E-binding protein. Nat Struct Biol 4: 717–724

Mayberry LK, Dennis MD, Allen ML, Nitka KA, Murphy PA, Campbell

L, Browning KS (2007) Expression and purification of recombinant

wheat translation initiation factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F,

eIF(iso)4F and eIF5. Methods Enzymol (in press)

McKendrick L, Morley SJ, Pain VM, Jagus R, Joshi B (2001) Phosphory-

lation of eukaryotic initiation factor 4E (eIF4E) at Ser209 is not required

for protein synthesis in vitro and in vivo. Eur J Biochem 268: 5375–5385

McKendrick L, Pain VM, Morley SJ (1999) Translation initiation factor 4E.

Int J Biochem Cell Biol 31: 31–35

Metz AM, Browning KS (1996) Mutational analysis of the functional

domains of the large subunit of the isozyme form of wheat initiation

factor eIF4F. J Biol Chem 271: 31033–31036

Metz AM, Timmer RT, Browning KS (1992) Isolation and sequence of a

cDNA encoding the cap binding protein of wheat eukaryotic protein

synthesis initiation factor 4F. Nucleic Acids Res 20: 4096

Michon T, Estevez Y, Walter J, German-Retana S, Le Gall O (2006) The

potyviral virus genome-linked protein VPg forms a ternary complex

with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E

affinity for a mRNA cap analogue 38. FEBS J 273: 1312–1322

Minich WB, Balasta ML, Goss DJ, Rhoads RE (1994) Chromatographic

resolution of in vivo phosphorylated and nonphosphorylated eukaryotic

translation initiation factor eIF-4E: increased cap affinity of the phos-

phorylated form. Proc Natl Acad Sci USA 91: 7668–7672

Moury B, Morel C, Johansen E, Guilbaud L, Souche S, Ayme V, Caranta C,

Palloix A, Jacquemond M (2004) Mutations in potato virus Y genome-

linked protein determine virulence toward recessive resistances in Cap-

sicum annuum and Lycopersicon hirsutum. Mol Plant Microbe Interact

17: 322–329

Muhandiram DR, Kay LE (1994) Gradient-enhanced triple-resonance

three-dimensional nmr experiments with improved sensitivity. J Magn

Reson B 103: 203–216

Naegele S, Morley SJ (2004) Molecular cross-talk between MEK1/2 and

mTOR signaling during recovery of 293 cells from hypertonic stress.

J Biol Chem 279: 46023–46034

Nicaise V, German-Retana S, Sanjuan R, Dubrana MP, Mazier M,

Maisonneuve B, Candresse T, Caranta C, LeGall O (2003) The eukar-

yotic translation initiation factor 4E controls lettuce susceptibility to the

potyvirus Lettuce mosaic virus. Plant Physiol 132: 1272–1282

Niedzwiecka A, Darzynkiewicz E, Stolarski R (2005) Deaggregation of

eIF4E induced by mRNA 5# cap binding. Nucleosides Nucleotides

Nucleic Acids 24: 507–511

Niedzwiecka A, Marcotrigiano J, Stepinski J, Jankowska-Anyszka M,

Wyslouch-Cieszynska A, Dadlez M, Gingras AC, Mak P, Darzynkiewicz E,

Sonenberg N, et al (2002) Biophysical studies of eIF4E cap-binding

protein: recognition of mRNA 5# cap structure and synthetic fragments

of eIF4G and 4E-BP1 proteins. J Mol Biol 319: 615–635

Orton KC, Ling J, Waskiewicz AJ, Cooper JA, Merrick WC, Korneeva

NL, Rhoads RE, Sonenberg N, Traugh JA (2004) Phosphorylation of

Mnk1 by caspase-activated Pak2/gamma-PAK inhibits phosphorylation

and interaction of eIF4G with Mnk. J Biol Chem 279: 38649–38657

Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data

collected in oscillation mode. Methods Enzymol 276: 307–326

Perrakis A, Sixma TK, Wilson KS, Lamzin VS (1997) wARP: improvement

and extension of crystallographic phases by weighted averaging of

multiple-refined dummy atomic models. Acta Crystallogr D Biol

Crystallogr 53: 448–455

Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, Agol

VI, Hellen CUT (2001) Molecular mechanisms of translation initiation

in eukaryotes. Proc Natl Acad Sci USA 98: 7029–7036

Pestova TV, Lomakin IB, Lee JH, Choi SK, Dever TE, Hellen CUT (2000)

The joining of ribosomal subunits in eukaryotes requires eIF5B. Nature

403: 332–335

Post CB (2003) Exchange-transferred NOE spectroscopy and bound ligand

structure determination. Curr Opin Struct Biol 13: 581–588

Prevot D, Darlix JL, Ohlmann T (2003) Conducting the initiation of protein

synthesis: the role of eIF4G. Biol Cell 95: 141–156

Pyronnet S, Dostie J, Sonenberg N (2001) Suppression of cap-dependent

translation in mitosis. Genes Dev 15: 2083–2093

Pyronnet S, Imataka H, Gingras AC, Fukunaga R, Hunter T, Sonenberg N

(1999) Human eukaryotic translation initiation factor 4G (eIF4G) re-

cruits Mnk1 to phosphorylate eIF4E. EMBO J 18: 270–279

Raught B, Gingras AC (1999) eIF4E activity is regulated at multiple levels.

Int J Biochem Cell Biol 31: 43–57

Read RJ (1986) Improved Fourier coefficients for maps using phases from

partial structures with errors. Acta Crystallogr A 42: 140–149

Redondo E, Krause-Sakate R, Yang SJ, Lot H, Le GO, Candresse T (2001)

Lettuce mosaic virus pathogenicity determinants in susceptible and

tolerant lettuce cultivars map to different regions of the viral genome.

Mol Plant Microbe Interact 14: 804–810

Reiling JH, Doepfner KT, Hafen E, Stocker H (2005) Diet-dependent

effects of the Drosophila Mnk1/Mnk2 homolog Lk6 on growth via

eIF4E. Curr Biol 15: 24–30

Ren JH, Goss DJ (1996) Synthesis of a fluorescent 7-methylguanosine

analog and a fluorescence spectroscopic study of its reaction with wheat

germ cap binding proteins. Nucleic Acids Res 24: 3629–3634





Richter JD, Sonenberg N (2005) Regulation of cap-dependent translation

by eIF4E inhibitory proteins. Nature 433: 477–480

Robaglia C, Caranta C (2006) Translation initiation factors: a weak link in

plant RNA virus infection. Trends Plant Sci 11: 40–45

Rodriguez CM, Freire MA, Camilleri C, Robaglia C (1998) The Arabidopsis

thaliana cDNAs coding for eIF4E and eIF(iso)4E are not functionally

equivalent for yeast complementation and are differentially expressed

during plant development. Plant J 13: 465–473

Ruffel S, Dussault MH, Palloix A, Moury B, Bendahmane A, Robaglia C,

Caranta C (2002) A natural recessive resistance gene against potato

virus Y in pepper corresponds to the eukaryotic initiation factor 4E

(eIF4E). Plant J 32: 1067–1075

Ruffel S, Gallois JL, Lesage ML, Caranta C (2005) The recessive potyvirus

resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E

gene. Mol Genet Genomics 274: 346–353

Ruffel S, Gallois JL, Moury B, Robaglia C, Palloix A, Caranta C (2006)

Simultaneous mutations in translation initiation factors eIF4E and

eIF(iso)4E are required to prevent pepper veinal mottle virus infection

of pepper. J Gen Virol 87: 2089–2098

Ruud KA, Kuhlow C, Goss DJ, Browning KS (1998) Identification and

characterization of a novel cap-binding protein from Arabidopsis

thaliana. J Biol Chem 273: 10325–10330

Sato M, Nakahara K, Yoshii M, Ishikawa M, Uyeda I (2005) Selective

involvement of members of the eukaryotic initiation factor 4E family in

the infection of Arabidopsis thaliana by potyviruses. FEBS Lett 579:

1167–1171

Scheper GC, Van Kollenburg B, Hu JZ, Luo YJ, Goss DJ, Proud CG (2002)

Phosphorylation of eukaryotic initiation factor 4E markedly reduces its

affinity for capped mRNA. J Biol Chem 277: 3303–3309

Schmitt TH, Zheng Z, Jardetzky O (1995) Dynamics of tryptophan binding

to Escherichia coli Trp repressor wild type and AV77 mutant: an NMR

study. Biochemistry 34: 13183–13189

Sha M, Wang YH, Xiang T, Van Heerden A, Browning KS, Goss DJ (1995)

Interaction of wheat germ protein synthesis initiation factor eIF-(iso)4F

and its subunits p28 and p86 with m7GTP and mRNA analogues. J Biol

Chem 270: 29904–29909

Sharma D, Rajarathnam K (2000) 13C NMR chemical shifts can predict

disulfide bond formation. J Biomol NMR 18: 165–171

Slepenkov SV, Darzynkiewicz E, Rhoads RE (2006) Stopped-flow kinetic

analysis of eIF4E and phosphorylated eIF4E binding to cap analogs and

capped oligoribonucleotides: evidence for a one-step binding mecha-

nism. J Biol Chem 281: 14927–14938

Sonenberg N, Dever TE (2003) Eukaryotic translation initiation factors and

regulators. Curr Opin Struct Biol 13: 56–63

Stein N, Perovic D, Kumlehn J, Pellio B, Stracke S, Streng S, Ordon F,

Graner A (2005) The eukaryotic translation initiation factor 4E confers

multiallelic recessive Bymovirus resistance in Hordeum vulgare (L.).

Plant J 42: 912–922

Tomoo K, Shen X, Okabe K, Nozoe Y, Fukuhara S, Morino S, Ishida T,

Taniguchi T, Hasegawa H, Terashima A, et al (2002) Crystal structures

of 7-methylguanosine 5#-triphosphate (m7GTP)- and P1-7-methylgua-

nosine-P3-adenosine-5#,5#- triphosphate (m7GpppA)-bound human

full-length eukaryotic initiation factor 4E: biological importance of the

C-terminal flexible region. Biochem J 362: 539–544

Tomoo K, Shen X, Okabe K, Nozoe Y, Fukuhara S, Morino S, Sasaki M,

Taniguchi T, Miyagawa H, Kitamura K, et al (2003) Structural features

of human initiation factor 4E, studied by x-ray crystal analyses and

molecular dynamics simulations. J Mol Biol 328: 365–383

Ueda H, Maruyama H, Doi M, Inoue M, Ishida T, Morioka H, Tanaka T,

Nishikawa S, Uesugi S (1991) Expression of a synthetic gene for human

cap binding protein (human IF-4E) in Escherichia coli and fluorescence

studies on interaction with mRNA cap structure analogues. J Biochem

(Tokyo) 109: 882–889

Vagin A, Teplyakov A (1997) MOLREP: an automated program for molec-

ular replacement. J Appl Crystallogr 30: 1022–1025

Van Heerden A, Browning KS (1994) Expression in Escherichia coli of the

two subunits of the isozyme form of wheat germ protein synthesis

initiation factor 4F: purification of the subunits and formation of an

enzymatically active complex. J Biol Chem 269: 17454–17457

Volpon L, Osborne MJ, Topisirovic I, Siddiqui N, Borden KL (2006) Cap-

free structure of eIF4E suggests a basis for conformational regulation by

its ligands. EMBO J 25: 5138–5149

Von der Haar T, Oku Y, Ptushkina M, Moerke N, Wagner G, Gross JD,

McCarthy JE (2006) Folding transitions during assembly of the eukar-

yotic mRNA cap-binding complex. J Mol Biol 356: 982–992

Waskiewicz AJ, Flynn A, Proud CG, Cooper JA (1997) Mitogen-activated

protein kinases activate the serine/threonine kinases Mnk1 and Mnk2.

EMBO J 16: 1909–1920

Waskiewicz AJ, Johnson JC, Penn B, Mahalingam M, Kimball SR, Cooper

JA (1999) Phosphorylation of the cap-binding protein eukaryotic trans-

lation initiation factor 4E by protein kinase Mnk1 in vivo. Mol Cell Biol

19: 1871–1880

Wishart DS, Bigam CG, Yao J, Abildgaard F, Dyson HJ, Oldfield E,

Markley JL, Sykes BD (1995) 1H, 13C and 15N chemical shift referencing

in biomolecular NMR. J Biomol NMR 6: 135–140

Worch J, Tickenbrock L, Schwable J, Steffen B, Cauvet T, Mlody B, Buerger H,

Koeffler HP, Berdel WE, Serve H, et al (2004) The serine-threonine kinase

MNK1 is post-translationally stabilized by PML-RARalpha and regu-

lates differentiation of hematopoietic cells. Oncogene 23: 9162–9172

Yoshii M, Nishikiori M, Tomita K, Yoshioka N, Kozuka R, Naito S, Ishikawa

M (2004) The Arabidopsis cucumovirus multiplication 1 and 2 loci

encode translation initiation factors 4E and 4G. J Virol 78: 6102–6111

Monzingo et al.




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